A Family of Highly Selective Allosteric Modulators of the Metabotropic Glutamate Receptor Subtype 5

Abstract

We have identified a family of highly selective allosteric modulators of the group I metabotropic glutamate receptor subtype 5 (mGluR5). This family of closely related analogs exerts a spectrum of effects, ranging from positive to negative allosteric modulation, and includes compounds that do not themselves modulate mGluR5 agonist activity but rather prevent other family members from exerting their modulatory effects. 3,3Ј-Difluorobenzaldazine (DFB) has no agonist activity, but it acts as a selective positive allosteric modulator of human and rat mGluR5. DFB potentiates threshold responses to glutamate, quisqualate, and 3,5-dihydroxyphenylglycine in fluorometric Ca 2ϩ assays 3-to 6-fold, with EC 50 values in the 2 to 5 M range, and at 10 to 100 M, it shifts mGluR5 agonist concentration-response curves approximately 2-fold to the left. The analog 3,3Ј-dimethoxybenzaldazine (DMeOB) acts as a negative modulator of mGluR5 agonist activity, with an IC 50 of 3 M in fluorometric Ca 2ϩ assays, whereas the analog 3,3Ј-dichlorobenzaldazine (DCB) does not exert any apparent modulatory effect on mGluR5 activity. However, DCB seems to act as an allosteric ligand with neutral cooperativity, preventing the positive allosteric modulation of mGluRs by DFB as well as the negative modulatory effect of DMeOB. None of these analogs affects binding of [ 3 H]quisqualate to the orthosteric (glutamate) site, but they do inhibit [ 3 H]3-methoxy-5-(2-pyridinylethynyl)pyridine binding to the site for 2-methyl-6-(phenylethynyl)-pyridine, a previously identified negative allosteric modulator. With the use of these compounds, we provide evidence that allosteric sites on GPCRs can respond to closely related ligands with a range of pharmacological activities from positive to negative modulation as well as to neutral competition of this modulation.Metabotropic glutamate receptors (mGluRs) are G proteincoupled receptors that bind glutamate to exert a modulatory influence on neuronal excitability and synaptic transmission in the central nervous system. The eight known members of the mGluR subfamily have been divided into three groups on the basis of their sequence identity, pharmacology, and preferred signal transduction mechanism. Group I mGluRs (mGluRs 1 and 5) are primarily localized postsynaptically where they modulate ion channel activity and neuronal excitability. The group I mGluRs are coupled to G ␣q and its associated effectors, such as phospholipase C. Groups II (mGluRs 2 and 3) and III (mGluRs 4, 6, 7, and 8) are primarily located presynaptically and regulate the release of neurotransmitters, including glutamate. Group II and III mGluRs are coupled to G ␣i and its associated effectors, such as adenylate cyclase. These latter two groups are distinguished from each other by their pharmacology; selective agonists and antagonists have been identified for each group (Conn and Pin, 1997). All mGluR subtypes possess a large (ϳ560 amino acids) extracellular amino-terminal domain that contains the glutamate agonist binding site. Thus, in...

Endogenous activation of metabotropic glutamate receptors supports the proliferation and survival of neural progenitor cells

“…The use of positive allosteric modulators of mGlu5 receptors may help to overcome these limitations. 39 The role of mGlu3 receptors in the regulation of NPC proliferation and survival is less clear. In culture, selective blockade of these receptors with nanomolar concentrations of LY341495 25 reduced NPC proliferation and survival.…”

An Automated System for the Analysis of G Protein-Coupled Receptor Transmembrane Binding Pockets: Alignment, Receptor-Based Pharmacophores, and Their Application

“…In agreement with the mutational analysis experiments, the orientation of MPEP includes interactions with the aromatic network of Y658 3.40 , W784 6.48 , and F787 6.51 and a hydrogen bond with T780 6.44 , which could prevent the movement of TM6 relative to TM3 and stabilize the inactive conformation of the receptor. Since DFB partially displaces the binding of the allosteric inhibitor [ 3 H]MPEP 61 and the DFB enhancer effect was completely absent in T780 6.44 A mutant receptors, T780 6.44 might serve as a polar anchor for both modulators. A mutation of W784 6.48 , resulting, on one hand, in a dramatic increase of the DFB-mediated potentiation and, on the other hand, in a loss of MPEP-mediated inhibition, supports the hypothesis that W784 6.48 plays a central role in controlling the activation states of the receptor.…”

Progress toward Positive Allosteric Modulators of the Metabotropic Glutamate Receptor Subtype 5 (mGlu5)

“…34,35 The discovery of the first selective mGlu 5 PAMs as one mechanism to begin to test the NMDA receptor hypofunction hypothesis were first described in 2003 by researchers at Merck beginning with a benzaldazine class of PAMs represented by 3,3 0 -difluorobenzaldazine (DFB, 1, Figure 1). 36 Through radioligand binding assays, this class of modulators was confirmed to interact at a single well characterized negative allosteric modulator binding site in a competitive manner, known as the MPEP-site. 36 Interestingly, from the very outset, these novel allosteric modulators displayed an unexpected and subtle capacity to modulate calcium mobilization with all three modalities of pharmacology, including positive and negative (DMeOB, 2) allosteric modulation as well as neutral or silent (DCB, 3) modulation through the use of simple halogen and alkoxy group substitution.…”

“…36 Through radioligand binding assays, this class of modulators was confirmed to interact at a single well characterized negative allosteric modulator binding site in a competitive manner, known as the MPEP-site. 36 Interestingly, from the very outset, these novel allosteric modulators displayed an unexpected and subtle capacity to modulate calcium mobilization with all three modalities of pharmacology, including positive and negative (DMeOB, 2) allosteric modulation as well as neutral or silent (DCB, 3) modulation through the use of simple halogen and alkoxy group substitution. This phenomena, referred to as a "molecular switch", is the result of a subtle molecular modification leading to gross changes in receptor pharmacology-response via allosteric modulation.…”